The dopamine transporter (DAT) exhibits several ionic currents that are either coupled to or uncoupled from the transport of substrate. Second messenger systems have been shown to modulate dopamine (DA) transport, however, the modulation of DAT-associated currents has not been studied in depth. Using the two-electrode voltage-clamp method to record from Xenopus oocytes expressing the human DAT, we examined the effects of arachidonic acid (AA) on membrane currents. AA (10-100 microM) stimulates a novel nonselective cation conductance seen only in oocytes expressing human DA transporter (hDAT). The AA-stimulated conductance is up to 50-fold greater than the current normally elicited by DA, but does not appear to arise from the modulation of previously described hDAT conductances, including the leak current and the current associated with electrogenic transport. In addition, DA dramatically potentiates and cocaine blocks the AA-stimulated DAT current. DA potentiates the AA-induced currents in the absence of sodium and chloride, indicating that these currents arise from processes distinct from those associated with substrate transport. The effects of AA were mimicked by other fatty acids with a rank order of potency correlated with their degree of unsaturation, suggesting that AA directly stimulates the novel cation current. Therefore, AA stimulation of this DAT-associated conductance may provide a novel mechanism for modulation of neuronal signaling.